Lipids

(Definition, General Properties, Functions) 

(Part of Q 1 + 2) 

* Definition- The cellular components that are soluble in organic solvents but sparingly soluble in water

* General Properties- 

$ One of the four major groups of biomolecules but unlike proteins, nucleic acids and polysaccharides they aren't polymers 

$ They are chemically the most diverse group

$ A catch-all category of things that are hydrophobic and sparingly soluble in water 

* Functions-

$ Form bilayers shich along with proteins are major components of membranes

 $ TAGs (triacyglycerols) are energy stores (fats and oils)

$ Many other functions as well: anitioxidants, Vitamin E, electron transporters, precursor for bile (break down fats in diet)

Question 1

I.                    Lipids

a.       Biological lipids are a chemically diverse group of coupounds, the common and defining feature of which is their insolubility in water

b.      Fats and oils are the principal stored forms of energy in many organisms

c.       Phospholipids and sterols are major structural elements of biological membranes.  These membranes have a bilayer formed by clustered phospholipids, forced together by hydrophobic interactions

d.      Other lipids play crucial roles as enzyme cofactors, electron carriers, light-absorbing pigments, hydrophobic anchors for proteins, “chaperones” to help membrane proteins fold, emulsifying agents in the digestive tract, hormones and intracellular messengers.

e.      The fats and oils used almost universally as stored forms of energy in living organisms are derivatives of fatty acids.

II.                  Amino Acids

a.       Form proteins via amino acid residues, linked by peptide bonds

b.      Some functions include: structure, catalysis (breakdown of complex things to simple things), signaling and transport

III.                Monosaccharides

a.       Monosaccharides linked by glycosidic bonds form carbohydrates

b.      Some functions include: energy, structure, cell recognition (sometimes carbs are tagged on a transported (forming) protein so that different structures can recognize the protein)

IV.                Nucleotides

a.       Nucleotide residues linked by phosphodiester bonds form nucleic acids

b.      They are important in information and energy carrier (NTP)

c.       Nucleotides have a variety of roles in cellular metabolism.  They are the energy currency in metabolic transactions, the essential chemical links the response of cells to hormones and other extracellular stimuli, and the structural components of an array of enzyme cofactors and metabolic intermediates.

d.      They are also the components of the nucleic acids DNA and RNA, the molecular repositories of genetic information

e.      The structure of every protein, and ultimately every biomolecule and cellular component, is a product of information programmed into the nucleotide sequence of a cell’s nucleic acids

Question 2     

(Part I-Definition of Lipids) 

Lipids are used in many different functions.  Such functions are the formation of lipid bilayers, energy storage and use, and signaling.  Signaling is important for hormones (epinephrine and insulin) along with other processes (regulation of transcription, glycogen breakdown, glucose release in bloodstream, cAMP stimulation/inhibition, nerve signals and muscle contractions, enzyme regulation, channel regulation, expression of specific genes (transcription), altering interaction through conformational changes (adhesion receptor) )

Question 2

(Part II-Contrasting Lipids with Proteins) 

When comparing lipids to proteins, a noticeable difference is that proteins are the molecular instruments through which genetic information is expressed.  They are used to make virtually everything in the body.  They are the tiniest building block.   One example is that proteins are polymers of amino acids, and amino acids are used to make genetic information and for many other functions.

Question 2

(Part III-Lipids vs Monosaccharides) 

When comparing lipids to monosaccharides, an immediate difference is the amount of energy (ATP) that can be derived from lipids.  A lipid yields much more energy that a monosaccharide, but it is hard to break down lipids to form this energy, so the easier route, monosaccharide catabolism, are the basic structure used for energy production.  Lipids are stored because of this while monosaccharides are used up in the body.  Insoluble carbohydrate polymers serve as structural and protective elements in the cell walls of plants, bacteria and the connective tissue of animals.  More complex carbohydrate polymers covalently attached to proteins or lipids act as signals that determine the intracellular location or metabolic fate of hybrid molecules (glycoconjugates-a compound containing a carbohydrate component bound covalently to a protein or lipids, forming a glycoprotein or glycolipid).  In this way, lipids and carbohydrates can work together to make signals.  Lastly, carbohydrates are the most abundant molecules on earth.

Question 2

(Part IV-Lipids vs Nucleotides) 

Fatty Acids

(Questions 3,4,5) 

* Structure-

$ Carboyxlic acids with long hydrocarbon chains

$ Most fatty acids have an even number of carbons and are broken down in two carbon units 

* Saturated-

$ Solid at room temperature; high melting point

$ All carbons havge max number of hydrogens; no double bonds

 * Unsaturated-

$ Have double bonds; almost always "cis"

$ Mono/poly double bonds

$ Determines whether a fatty acid is solid or liquid

$ Liquid at room temperature; low melting point

* Properties-

$ Amphipathic

$ Form micelles in aqueous solutio

$ Most have enven number of carbons

$ Double bonds are almost always cis 

-Cis kinks the molecule

-Trans double bounds are rare in nature and don't kink the molecule--bad for cholesterol

$ Multiple double bonds ar ealmost always three carbons apart, i.e. not conjugated

* Nomenclature (need to know)

$ Common names, shorthand carbon skeletons, systemic names, omega fatty acids (start from methyl group)

$ Note 1- increasing carbon number increases the melting point

$ Note 2- increasing double bonds decreases the melting point 

Non-Membrane Lipids

(Question 6) 

a.       Triacylglycerols (TAGs/oils and fats)

                                                               i.      Structure-

1.       Simplest lipids constructed from fatty acids

2.       A glycerol backbone with three fatty acids attached by ester linkages

3.       The fatty acids can be the same but are most often different

4.       Simple: have the same fatty acids

5.       Complex: have different fatty acids

b.      Properties-

                                                               i.      Most abundant  lipids in the human body but DO NOT form membranes

                                                             ii.      Hydrophobic-stored in an anhydrous form

                                                            iii.      Fatty acids attached determine properties (liquid or solid at room temperature)

                                                           iv.      Highly reduced carbons have a lot more energy

                                                             v.      TAGs are unhydrated so the organism that carries fat as a fuel does not have to carry the extra weight of water of hydration that is associated with stored polysaccharides

c.       Functions-

                                                               i.      Primarily metabolic storage molecules (fats in animals, oils in plants)

1.       Provide ~6 times the metabolic energy of carbohydrates

2.       Glycogen stores last 24 hours –TAG stores last 2-3 months

3.       Provide insulation

4.       Normal man is 21% fat, normal woman is 26% fat

Membrane Lipids

(Part I-Introduction, Question 7) 

Key Concept:

-Various polar head groups

-Various non-polar tails

 -Properties of nonpolar portions determine the properties of the lipid incorporated into membrane proteins

* General Structure-

$ Polar head group

$ Hydrophobic portion made up of fatty acids

* General Properties

$ Membrane lipids are amphipathic

$ Form bilayers and liposomes in aqueous solutions

Membrane Lipids

Glycerophospholipids

(Q 7) 

* Structure-

$ Glycerol backbone with fatty acids esterified to C1 and C2 and a head group attached to C3 by a phosphodiester linkage

$ Some have ether linkage to C1

$ enormous diversity of structure

* Properties-

$ Different fatty acids alter properties

$ C1=often saturated

$ C2=often mono- or polyunsaturated

$ Different head-groups alter properties

* Function-

$ Major membrane constituent

$ Precursor for signal molecules 

* Nomenclature-see fig 10-8

Glycerophospholipids

(Book Notes) 

I.                   Glycerophospholipids Are Derivatives of Phosphatidic Acid

a.       Glycerophospholipids, also called phosphoglycerides, are membrane lipids in which two fatty acids are attached in ester linkage to the first and second carbons of glycerol, and a highly polar or charged group is attached through a phosphodiester linkage to the third carbon.  Glycerol is prochiral; it has no asymmetric carbons, but attachment of phosphate at one end converts it into a L-glycerol 3-phosphate

b.      In all these compounds (glycerophospholipids) the head group is joined to glycerol through a phosphodiester bond, in which the phosphate group bears a negative charge at neutral pH

c.       The fatty acids in glycerophospholipids can be any of a wide variety, so a given phospholipids may consist of a number of fatty acids

Membrane Lipids

Galactolipids and Sulfolipids 

(Q 7) 

* Structure-

$ Glycerol backbone with fatty acids esterfied to C1 and C2 and a head group attached to C3 by a glycosidic linkage

* Properties-

$ Similar to glycerophospholipids

* Function-

$ Abundant in plant chloroplasts

$ Most abundant membrane lipids on the planet

$ May compensate for phosphate limitation in plants

* Examples-

@ Monogalactosyldiacylglycerol (MGDG)

@ Digalactosyldiacylglycerol (DGDG)

-major component of chloroplast membranes

@ DAG-diacylglycerol

-two fatty acids attached

-signaling molecule

Galactolipids and Sulfolipids

(Book Notes) 

I.                   Chloroplasts Contain Galactolipids and Sulfolipids

a.       The second group of membrane lipids are those that predominate in plant cells: the galactolipids, in which one or two galactose residues are connected by a glycosidic linkage to C-3 of a 1,2-diacylglycerol

b.      Galactolipids are localized in the thylakoid membranes (internal membranes) of chloroplasts, they make up 70-80% of the total membrane lipids of a vascular plant.  They are probably the most abundant membrane lipids in the biosphere

c.       Plant membranes also contain sulfolipids, in which a sulfonated glucose residue is joined to a diacylglycerol in glycosidic linkage.

d.      In sulfolipids, the sulfonate on the head group bears a fixed negative charge like that of the phosphate group in phospholipids

Membrane Lipids 

Sphingolipids

(Q 7) 

* Structure-

$ Built on the 16 to 20 carbon amino alcohol sphingosine

-Amino group on C2

-Hydroxyl group on C3 

-Trans double bond

$ A fatty acid is attached by an amide bond to the nitrogen on carbon 2 of sphingosine backbone with a polar head group attached to C1

 * Properties-

$ Similar to glycerophopholipids and galactolipids

* Function-

$ Prevalent in myelin and neuronal membranes

$ Cell recognition (ABO blood groups=globosides) 

Sphingolipids

(Book Notes) 

I.                   Sphingolipids Are Derivatives of Sphingosine

a.       Sphingolipids, the fourth large class of membrane lipids, also have a polar head group and two nonpolar tails, but unlike glycerophospholipids and galactolipids they contain no glycerol

b.      Sphingolipids are composed of one molecule of the long-chain amino alcohol sphingosine or one of its derivatives, one molecule of a long-chain fatty acid, and a polar head group that is joined by a glycosidic linkage in some cases and by phosphodiester in others

c.       Carbons C-1, C-2, and C-3 of the sphingosine molecule are structurally analogous to the three carbons of glycerol in glycerophospholipds.  When a fatty acid is attached in amide linkage to the –NH2 on C-2, the resulting compound is a ceramide, which is structurally similar to a diacylglycerol.  Cermaide is the structural parent of all sphingolipids

d.      There are three subclasses of sphingolipids, all derivatives of ceramide but differing in their head groups:

                                                              i.      Sphingomyelins-

1.      contain phosphocholine or phosphoethanolamine as their polar head group and are therefore classified along with glycerophospholipids as phospholipids

2.      They resemble phosphatidylcholines in their general properties and three-dimensional structure, and in having no net charge on their head groups

3.      Sphingomyelins are present in the plasma membranes of animal cells and are especially prominent in myelin, a membranous sheath that surrounds and insulates the axons of some neurons-thus the name “spingomyelins”

                                                            ii.      Glycosphingolipids-

1.      Occur largely in the outer face of plasma membranes, have head groups with one or more sugars connected directly to the –OH at C-1 of the ceramide moiety; they do not contain phosphate

2.      Cerebrosides have a single sugar linked cermainde; those with galactose are characteristically found in the plasma membranes of cells in neural tissue, and those with glucose in the plasma membranes of cells in nonneural tissues. 

3.      Globosides are neutral glycosphingolipids with two or more sugars

                                                          iii.      Gangliosides-

1.      Are the most complex sphingolipids, have oligosaccharides as their polar head groups and one or more residues of N-acetylneuraminic acid, a sialic acid, at the termini. 

2.      Sialic acid gives gangliosides the negative charge at pH 7 that distinguishes them from glycosides

Membrane Lipids

Cholesterol

(Q 7) 

* Structure-

$ Rigid, 4 fused rings, planar structure

$ Hydrocarbon Tail

$ OH on C3

$ Built from Isoprene-no fatty acids

* Properties-

$ Amphipathic

- Hydrocarbon rings and tail are hydrophobic

-OH is polar 

$ Rigid

* Functions-

$ Membrane fluidity

$ Signal precursor (steroid hormones)

$ Bile acid precursor (emulsifiers for digestion) 

Sterols and Cholesterol

(Book Notes) 

I.                   Sterols-

a.       Structural lipids present in the membranes of most eukaryotic cells

b.      The characteristic structure of this fifth group of membrane lipids is the steroid nucleus, consisting of fused rings, three with six carbons and one with five

c.       The sterol nucleus is almost planar and relatively rigid

d.      The fused rings do not allow rotation about the C-C bonds

II.                Cholesterol

a.       The major sterol group in animal tissues, is amphipathic with a polar head group and a nonpolar hydrocarbon body

b.      The sterols of all eukaryotes are synthesized from simple five carbon isoprene subunits

c.       They are membrane constituents and serve as precursors for a variety of products with specific biological activities

Question 8

(need to work on still)     

Phopholipases

(Question 9) 

* Phospholipases-enzymes that hydrolyze bonds in phospholipids (turn over lipids)

* Specificity-

$ Phospholipase A1 (PLA1) cleaves between fatty acid and carbon 1 of glycerol

$ Phospholipase A2 (PLA2) cleaves between fatty acid and carbon 2 of glycerol

$ Phospholipase C (PLC) cleaves between carbon 3 of glycerol and phosphate

$ PHospholipase D (PLD) cleaves between phosphate and head group

* Functions-

$ Digestion-cleaving lipids

$ Lipid turn over

$ Signaling

Key Concept: Metabolism of lipids does a number of things but can also generate signals in cells 

Phospholipases

(Book Notes) 

I.                   Specific Hydrolysis Aids in Determination of Lipid Structure

a.       Certain classes of lipids are susceptible to degradation under specific conditions

b.      Enzymes that specifically hydrolyze certain lipids are also useful in the determination of lipid structure.  Phospholipases A, C, and D each split particular bonds in the phospholipids and yield products with characteristic solubilities and chromatographic behaviors

c.       Phospholipase C, for example, releases a water-soluble phosphoryl alcohol and a chloroform-soluble diacylglycerol, each of which can be characterized separately to determine the structure of the intact phospholipids

Lipid Signaling

(Question 10) 

*Lipid Signaling-hydrolysis products of membrane lipids

* PLC-

$ PIP2 is in inner leaf of plasma membrane

$ PLC activated by hormones

$ Releases IP3 and DAG, both of which are signals

 $ View fig 10-17

*PLA2-

$ Releases omega-3 and -6 fatty acids like arachidonate, EPA or DHA which are processed to eicosanoids (see next card for definition) 

- Local (paracrine) hormones

- Site of action localized and very brief to half life (seconds to minutes)

* Leukotriene-

 $ Control of smooth muscle contractions is regulated by leukotrienes

$ Derived from arachidonate

$ Short half-lives, very localized

$ Asthma

* Thromboxanes-

$ Involved in inducing blood clots

$ Not dependent on cycloxygenase 

* Prostaglandins

$ Involved in fever, inflammation 

* Eicosanoids are paracrine hormones, substances that act only on cells near the point of hormone synthesis instead of being transported in the blood to act on cells in other tissues or organs. 

 * They are known to be involved in reproductive function; in the inflammation, fever and pain associate with injury or disease; in the formation of blood clots and the regulation of blood pressure, in gastric acid secretion; and in a variety of other processes importnat in human health or disease

* All eicosanoids are derived from arachidonic acid, the 20-carbon polyunsaturated fatty acid from which they take their general name.  There are three classes of eicosanoids: prostaglandins, thromboxanes, and leukotrienes 

I.                   Eicosanoids Are Formed from 20-Carbon Polyunsaturated Fatty Acids

a.       Eicosanoids are a family of very potent biological signaling molecules that act as short-range messengers, affecting tissues near the cells that produce them

b.      In response to hormonal or other stimuli, phospholipase A2, present in most types of mammalian cells, attacks membrane phospholipids, releasing arachidonate from the middle carbon of glycerol

c.       Enzymes of the smooth ER then convert arachidonate to prostaglandins, beginning with the formation of prostaglandin H2, the immediate precursors of many other prostaglandins and of thromboxanes

d.      The two reactions that lead to PGH2 are catalyzed by a bifunctional enzymes (COX).  P 800 [aspirin]

e.       Thromboxane synthase, present in blood platelets (thrombocytes), converts PGH2 to thromboxane A2, from which other thromboxanes are derived. 

f.       Thromboxanes induce constriction of blood vessels and platelet aggregation, early steps in blood clotting.  Low doses of aspirin, taken regularly, reduce the probability of heart attacks and strokes by reducing thromboxane production

g.      Leukotrienes are linear compounds whose synthesis begins with the action of several lipoxygenases that catalyzed the incorporation of molecular oxygen into arachidonate.  These enzymes, found in leukocytes and in heart, brain, lung and spleen, are mixed-function oxidases that use cytochrome p-450. 

h.      The various leukotrienes differ in the position of the peroxide group introduced by the lipoxygenases.  This linear pathway from arachindonate, unlike the cyclic pathway, is not inhibited by aspirin or other NSAIDs.

             I.      Thromboxanes:

a.       Have six-membered ring containing an ether they are produced by platelets (also called thrombocytes) and act in the formation of blood clots and the reduction of blood flow to the site of a clot

          II.      Leukotrienes:

a.       First found in leukocytes, contain three conjugated double bonds.  They are powerful biological signals

III.  Prostaglandins:

a. Contain a five-carbon ring originating from the chain of arachidonic acid

b. Some prostaglandins stimulate contraction of the smooth muscle of teh uterus during menstration and labor.  Others effect blood flow to specific organs, the wake-sleep cycle, and the responsiveness of certain tissues to hormones such as epinephrine and glucagon 

c. Prostaglandins in a third group elevate body temperature (producing fever) and cause inflammation and pain 

More on Lipid Signaling

(Q 10) 

(Needs some tweaking) 

* Hydrolysis products of membrane lipids

* PLA2:

$ Releases the omega-3 and -6s which are processed to eicosanoids

-COX function is required for prostaglandin and thromboxane formation

-COX is the site of action for non-steroidal anti-inflammatory drugs (NSAIDs): aspirin, Tylenol, ibuprofen, Celebrex, Vioxx

 * COX-1 and COX-2 are isozymes (~65% identical). 

$ COX-1 prodouces prostaglandins invovled in controlling gastric mucin secretion (keeps stomach from eating itself)

$ COX-2 produces prostaglandins invovled in pain, inflammation and fever.  Celebrex, Vioxx, etc. were designed based on differences in COX-1 and COX-2 crystal structures.  

-They bind ~1,000x better to COX-2, so greatly reduced stomach problems.  It also turns out they increase the risk of cardiovascular problems

* COX inhibition (not sure where to find this...wasn't in box in book)

-Irreversible

-Reversible

*Non-specific/specific 

Lipid Signaling

(Q 10) 

*Sterols-

$ Steroids are endocring hormones

-Potent signaling molecules derived from cholesterol

-Circulate throughout the body

- Control diverse bodily functions

Question 11

(Needs tweaking) 

Working With Lipids 

(Question 12)

Working with Lipids

I.                   Because lipids are insoluble in water, their extraction and subsequent fractionation require the use of organic solvents and some techniques not commonly used in the purification of water-soluble molecules such as proteins and carbohydrates.  In general, complex mixtures of lipids are separated by differences in the polarity or solubility of the components in nonpolar solvents.

a.       Lipids that contain ester- or amide-linked fatty acids can be hydrolyzed by treatment with acid or alkali or with highly specific hydrolytic enzymes (phospholipases, glycosidases) to yield their component parts for analysis

II.                Lipid Extraction Requires Organic Solvents

a.       Neutral lipids (triaxyglycerolds, waxes, pigments and so forth) are readily extracted from tissues with ethyl ether, choloroform, or benzene, solvents that do not permit lipid clustering driven by hydrophobic interactions

b.      Membrane lipids are more successfully extracted by more polar organic solvents, such as ethanol or methanol, which reduce the hydrophobic interactions among lipids molecules while also weakening the hydrogen bonds and electrostatic interactions that bind membrane lipids to membrane proteins

c.       A commonly used extractant is a mixture of chloroform, methanol, and water, initially in volume proportions that are miscible, producing a single phase. 

                                                              i.      After tissue is homogenized in this solvent to extract all lipids, more water is added to the resulting extract and the mixture separates into two phases, methanol/water (top phase) and chloroform (bottom phase)

                                                            ii.      The lipids remain in the chloroform layer, and more polar molecules such as proteins and sugars partition into the methanol/water layer

III.             Adsorption Chromatography Separates Lipids of Different Polarity

a.       Complex mixtures of tissue lipids can be fractionated by chromatographic procedures based on the different polarities of each class of lipid

b.      In adsorption chromatography, an insoluble, polar material such as silica gel is packed into a glass column and the lipid mixture is applied to the top of the column

c.       The polar lipids bind tightly to the polar silicic acid, but the neutral lipids pass directly through the column and emerge in the first chloroform wash.  The polar lipids are then eluted, in order of increasing polarity by washing the column with solvents of progressively higher polarity

d.      Uncharged but polar lipids (cerebrosides for example) are eluted with acetone and very polar or charged lipids (Such as glycerophospholipids) are eluted with methanol

IV.             Gas-Liquid Chromatography Resolves Mixtures of Volatile Lipid Derivatives

a.       Gas-liquid chromatography separates volatile components of a mixture according tot heir relative tendencies to dissolve in the inert material packed in the chromatography column and to volatize and move through the column, carried by a current of an inert gas such as helium.

b.      Some lipids are naturally volatile, but most must first be derivatized to increase their volatility (that is, lower their boiling point)

c.       For an analysis lipids are converted into their methyl esters.  These fatty acyl methyl esters are then loaded onto the gas-liquid chromatography column, and the column is heated to volatize the compounds

d.      Those fatty acyl esters most soluble in the column material partition into (dissolve in) that material; the less soluble lipids are carried by the stream of inert gas and emerge first from the column

e.       The order of elution depends on the nature of the solid adsorbant in the column and on the boiling point of the components of the lipid mixture.  Using these techniques, mixtures of fatty acids of various chain lengths and various degrees of unsaturation can be completely resolved

V.                Specific Hydrolysis Aids in Determination of Lipid Structure

a.       Certain classes of lipids are susceptible to degradation under specific conditions

b.      Enzymes that specifically hydrolyze certain lipids are also useful in the determination of lipid structure.  Phospholipases A, C, and D each split particular bonds in the phospholipids and yield products with characteristic solubilities and chromatographic behaviors

c.       Phospholipase C, for example, releases a water-soluble phosphoryl alcohol and a chloroform-soluble diacylglycerol, each of which can be characterized separately to determine the structure of the intact phospholipids

VI.             Mass Spectometry Reveals Complete Lipid Structure

a.       To establish unambiguously the length of a hydrocarbon chain or the position of double bonds, mass spectral analysis of lipids or their volatile derivatives is invaluable

b.      The chemical properties of similar lipids are very much alike, and their position of elution from the various chromatographic procedures often do not distinguish between them

c.       When the effluent from a chromatography column is sampled by mass spectrometry, however, the components of a lipid mixture can be simultaneously separated and identified by their unique patter of fragmentation

Summary 10.4 Working with Lipids

• In the determination of lipid composition, the lipids are first extracted from tissues with organic solvents and separated by thin-layer, or gas-liquid, or high-performance liquid chromatography
• Phospholipases specific for one of the bonds in a phoshpolipid can be used to generate simpler compounds for subsequent analysis
• Individual lipids are identified by their chromatographic behavior, their susceptibility to hydrolysis by specific enzymes, or mass spectrometry